【摘要】 细胞色素氧化酶(Cytochrome oxidase,COX)是线粒体内呼吸链电子传递的终末复合物,是线粒体氧化能力的关键调节物质。细胞色素C氧化酶亚基I(COXⅠ)是细胞色素C氧化酶具有酶催化活性的3个亚基之一。本研究以本实验室构建的青石斑鱼消减杂交cDNA文库中长587bp的EST序列为基础,采用RACE-PCR方法克隆鉴定出青石斑鱼(Epinephelus awoara)细胞色素C氧化酶亚基I基因。研究结果表明:青石斑鱼COXⅠ全长1659bp,5’端非编码区3bp,3’端非编码区105bp,开放阅读框1551bp,编码516个氨基酸。序列分析表明,青石斑鱼COXⅠ与线纹刺尾鲷COXⅠ同源性最高,达96.9%。采用半定量RT-PCR方法研究COXⅠ基因在青石斑鱼各组织中的表达特征,结果表明COXⅠ基因在正常的青石斑鱼和注射了副溶血弧菌灭活疫苗的青石斑鱼的脾、肝、心、头肾、肾、肌肉和鳃中都有转录表达。注射副溶血弧菌灭活疫苗后,除了鳃组织,其他组织中COXⅠ基因表达量较对照组都有显著提高(P<0.05),而鳃组织中的COXⅠ基因表达量没有显著变化。更多还原

【Abstract】 COX Ⅰ is the subunit one of the Cytochrome c oxidase which provides a critical function during respiration by transferring electrons from Cytochrome c to oxygen and contributing to ATP generation. A COX Ⅰ cDNA sequence (GenBank accession number: EU363802) was isolated and identified from the total RNA of Epinephelus awoara spleen using the combined methods of BD SMARTTM cDNA synthesis and RACE-PCR. The full length sequence of COXⅠ cDNA was 1 659 bp,containing 3 bp 5’UTR,105 bp 3’UTR and an open reading frame of 1 551 bp that encodes a protein composed of 516 amino acids. The deduced molecular weight was 56.0 kDa and the theoretical pI was 6.48. The amino acid sequence of E. awoara COXⅠ shared 86.5%-96.9% of residue identity with Homo sapines(86.5%), Mus musculus(88.7%),Danio rerio(94.2%) and Acanthurus lineatus(96.9%). Semi-quantitative RT-PCR analysis was used to study the tissue distribution and expression level of mRNA coding for COXⅠ and it showed that expression of COXⅠ could be detected in the spleen,heart,liver,anterior kidney,kidney,muscle and gill of the normal and the formalin-inactivated Vibrio Parahaemolyticus vaccine-challenged groups of E.awoara. Following vaccine-challenges, COXⅠ expression in all tissues except gill was up-regulated(P<0.05),compared with the normal E. awoara.更多还原